The most commonly used agents in the treatment of asthma are drugs that activate human beta2-adrenergic receptors (beta2ARs), leading to the relaxation of constricted airways. The surface expression of beta2ARs on cells is a key determinant of their signaling capacity in response to beta-agonists. After exposure to agonist, most beta2ARs rapidly traffic from the cell surface into early endosomes by clathrin-dependent endocytosis. Following this, beta2ARs may be sorted to either the recycling pathway, and thereby repopulate the cell surface with resensitized receptors, or be transported to lysosomes where they are degraded (a process termed downregulation). The mechanisms governing these sorting steps at the level of the early endosome are poorly understood, although the trafficking of beta2ARs to lysosomes and their subsequent downregulation probably requires receptor ubiquitination and involves proteasomes. Our long-range goal is to define the molecular machinery regulating these sorting steps and how the endosome sorting of beta2ARs modulates the surface expression and resensitization of receptors following agonist-triggered desensitization. For this application, we propose the following three specific aims: (1). determine if Hrs regulates rapid beta2AR recycling and resensitization through its interaction with Actinin-4/BERP/Myosin Vb (thereby forming the CART complex); (2). elucidate the role of the human Vps34-interacting protein, the rab7 GPTase, in the regulation of beta2AR recycling; (3). determine if the rab7-interacting protein, XAPC7, regulates beta2AR trafficking by linking proteasome function and the endocytic pathway. These aims will be accomplished using a combination of complementary molecular, biochemical, and advanced microscopic techniques. The proposed studies will be performed in HEK293 cells, and key findings will be confirmed in cultured human airway cells. Relevance: We expect that these studies will provide new targets for therapeutic interventions to increase the surface expression and resensitization (and therefore receptor signaling capacity) of beta2ARs following agonist stimulation and improve responsiveness to beta-agonists. This improved responsiveness may lead to reduced symptoms in patients with chronic asthma who use beta-agonists regularly and decreased hospitalizations and deaths from acute asthma exacerbations, where beta-agonists remain the first-line treatment. [unreadable] [unreadable] [unreadable]